The antibacterial activity of (2Z,3R,6S)-4-hydrazono-3,6-dimethyl-2-(3-methylbutylidene) octahydrobenzofuran-3-ol on Bacillus subtilis and Staphylococcus aureus were studied. The in vitro antibacterial circle and minimum inhibitory concentration (MIC) were used to evaluate its antibacterial effect. The results showed that the compound antibacterial circles (at 1 μg/mL) on Bacillus subtilis and Staphylococcus aureus were 19 ± 1.12 mm and 13 ± 0.94 mm; MIC of Bacillus subtilis was 0.0166 mg/mL. The novel compound has a strong antibacterial activity in vitro, especially on Bacillus subtilis.